Three-dimensional topography of scapular nutrient foramina.

PURPOSE: The aim of this study is to describe the number and location of the nutrient foramina in human scapulae which can minimize blood loss during surgery. METHODS: 30 cadaveric scapulae were macerated to denude the skeletal tissue. The nutrient foramina of 0.51 mm and larger were identified and labeled by adhering glass beads. CT scans of these scapulae were segmented resulting in a surface model of each scapula and the location of the labeled nutrient foramina. All scapulae were scaled to the same size projecting the nutrient foramina onto one representative scapular model. RESULTS: Average number of nutrient foramina per scapula was 5.3 (0-10). The most common location was in the supraspinous fossa (29.7%). On the costal surface of the scapula, most nutrient foramina were found directly inferior to the suprascapular notch. On the posterior surface, the nutrient foramina were identified under the spine of the scapula in a somewhat similar fashion as those on the costal surface. Nutrient foramina were least present in the peri-glenoid area. CONCLUSION: Ninety percent of scapulae have more than one nutrient foramen. They are located in specific areas, on both the posterior and costal surface.

Correction to: Three­dimensional topography of scapular nutrient foramina.

Correction to: Surgical and Radiologic Anatomy.

LSD1 Histone Demethylase Assays and Inhibition.

The lysine-specific demethylase (LSD1) is a flavin-dependent amine oxidase that selectively removes one or two methyl groups from histone H3 at the Lys4 position. Along with histone deacetylases 1 and 2, LSD1 is involved in epigenetically silencing gene expression. LSD1 has been implicated as a potential therapeutic target in cancer and other diseases. In this chapter, we discuss several approaches to measure LSD1 demethylase activity and their relative strengths and limitations for inhibitor discovery and mechanistic characterization. In addition, we review the principal established chemical functional groups derived from monoamine oxidase inhibitors that have been investigated in the context of LSD1 as demethylase inhibitors. Finally, we highlight a few examples of recently developed LSD1 mechanism-based inactivators and their biomedical applications.

The epigenetic regulators CBP and p300 facilitate leukemogenesis and represent therapeutic targets in acute myeloid leukemia.

Growing evidence links abnormal epigenetic control to the development of hematological malignancies. Accordingly, inhibition of epigenetic regulators is emerging as a promising therapeutic strategy. The acetylation status of lysine residues in histone tails is one of a number of epigenetic post-translational modifications that alter DNA-templated processes, such as transcription, to facilitate malignant transformation. Although histone deacetylases are already being clinically targeted, the role of histone lysine acetyltransferases (KAT) in malignancy is less well characterized. We chose to study this question in the context of acute myeloid leukemia (AML), where, using in vitro and in vivo genetic ablation and knockdown experiments in murine models, we demonstrate a role for the epigenetic regulators CBP and p300 in the induction and maintenance of AML. Furthermore, using selective small molecule inhibitors of their lysine acetyltransferase activity, we validate CBP/p300 as therapeutic targets in vitro across a wide range of human AML subtypes. We proceed to show that growth retardation occurs through the induction of transcriptional changes that induce apoptosis and cell-cycle arrest in leukemia cells and finally demonstrate the efficacy of the KAT inhibitors in decreasing clonogenic growth of primary AML patient samples. Taken together, these data suggest that CBP/p300 are promising therapeutic targets across multiple subtypes in AML.

Ghrelin.

BACKGROUND: The gastrointestinal peptide hormone ghrelin was discovered in 1999 as the endogenous ligand of the growth hormone secretagogue receptor. Increasing evidence supports more complicated and nuanced roles for the hormone, which go beyond the regulation of systemic energy metabolism. SCOPE OF REVIEW: In this review, we discuss the diverse biological functions of ghrelin, the regulation of its secretion, and address questions that still remain 15 years after its discovery. MAJOR CONCLUSIONS: In recent years, ghrelin has been found to have a plethora of central and peripheral actions in distinct areas including learning and memory, gut motility and gastric acid secretion, sleep/wake rhythm, reward seeking behavior, taste sensation and glucose metabolism.

Supracondylar osteotomy for the treatment of cubitus varus in children: a systematic review.

Cubitus varus is the most frequent complication following the treatment of supracondylar humeral fractures in children. We investigated data from publications reporting on the surgical management of cubitus varus found in electronic searches of Ovid/MEDLINE and Cochrane Library databases. In 894 children from 40 included studies, the mean age at initial injury was 5.7 years (3 to 8.6) and 9.8 years (4 to 15.7) at the time of secondary correction. The four osteotomy techniques were classified as lateral closing wedge, dome, complex (multiplanar) and distraction osteogenesis. A mean angular correction of 27.6º (18.5° to 37.0°) was achieved across all classes of osteotomy. The meta-analytical summary estimate for overall rate of good to excellent results was 87.8% (95% CI 84.4 to 91.2). No technique was shown to significantly affect the surgical outcome, and the risk of complications across all osteotomy classes was 14.5% (95% CI 10.6 to 18.5). Nerve palsies occurred in 2.53% of cases (95% CI 1.4 to 3.6), although 78.4% were transient. No one technique was found to be statistically safer or more effective than any other.

Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation.

BACKGROUND: Heat shock protein 90 (HSP90) has a key role in the maintenance of the cellular proteostasis. However, HSP90 is also involved in stabilisation of oncogenic client proteins and facilitates oncogene addiction and cancer cell survival. The development of HSP90 inhibitors for cancer treatment is an area of growing interest as such agents can affect multiple pathways that are linked to all hallmarks of cancer. This study aimed to test the hypothesis that targeting cysteine residues of HSP90 will lead to degradation of client proteins and inhibition of cancer cell proliferation. METHODS: Combining chemical synthesis, biological evaluation, and structure-activity relationship analysis, we identified a new class of HSP90 inhibitors. Click chemistry and protease-mass spectrometry established the sites of modification of the chaperone. RESULTS: The mildly electrophilic sulphoxythiocarbamate alkyne (STCA) selectively targets cysteine residues of HSP90, forming stable thiocarbamate adducts. Without interfering with the ATP-binding ability of the chaperone, STCA destabilises the client proteins RAF1, HER2, CDK1, CHK1, and mutant p53, and decreases proliferation of breast cancer cells. Addition of a phenyl or a tert-butyl group in tandem with the benzyl substituent at nitrogen increased the potency. A new compound, S-4, was identified as the most robust HSP90 inhibitor within a series of 19 derivatives. CONCLUSION: By virtue of their cysteine reactivity, sulphoxythiocarbamates target HSP90, causing destabilisation of its client oncoproteins and inhibiting cell proliferation.

The assessment of scapular radiographs: analysis of anteroposterior radiographs of the shoulder and the effect of rotational offset on the glenopolar angle.

The glenopolar angle assesses the rotational alignment of the glenoid and may provide prognostic information and aid the management of scapula fractures. We have analysed the effect of the anteroposterior (AP) shoulder radiograph rotational offset on the glenopolar angle in a laboratory setting and used this to assess the accuracy of shoulder imaging employed in routine clinical practice. Fluoroscopic imaging was performed on 25 non-paired scapulae tagged with 2 mm steel spheres to determine the orientation of true AP views. The glenopolar angle was measured on all the bony specimens rotated at 10° increments. The mean glenopolar angle measured on the bone specimens in rotations between 0° and 20° and thereafter was found to be significantly different (p < 0.001). We also obtained the AP radiographs of the uninjured shoulder of 30 patients treated for fractures at our centre and found that none fitted the criteria of a true AP shoulder radiograph. The mean angular offset from the true AP view was 38° (10° to 65°) for this cohort. Radiological AP shoulder views may not fully project the normal anatomy of the scapular body and the measured glenopolar angle. The absence of a true AP view may compromise the clinical management of a scapular fracture.

Discovery of entry inhibitors for HIV-1 via a new de novo protein design framework.

A new (to our knowledge) de novo design framework with a ranking metric based on approximate binding affinity calculations is introduced and applied to the discovery of what we believe are novel HIV-1 entry inhibitors. The framework consists of two stages: a sequence selection stage and a validation stage. The sequence selection stage produces a rank-ordered list of amino-acid sequences by solving an integer programming sequence selection model. The validation stage consists of fold specificity and approximate binding affinity calculations. The designed peptidic inhibitors are 12-amino-acids-long and target the hydrophobic core of gp41. A number of the best-predicted sequences were synthesized and their inhibition of HIV-1 was tested in cell culture. All peptides examined showed inhibitory activity when compared with no drug present, and the novel peptide sequences outperformed the native template sequence used for the design. The best sequence showed micromolar inhibition, which is a 3-15-fold improvement over the native sequence, depending on the donor. In addition, the best sequence equally inhibited wild-type and Enfuvirtide-resistant virus strains.

Delayed operative management of fractures of the scapula.

Between 1998 and 2007, 22 patients with fractures of the scapula had operative treatment more than three weeks after injury. The indications for operation included displaced intra-articular fractures, medialisation of the glenohumeral joint, angular deformity, or displaced double lesions of the superior shoulder suspensory complex. Radiological and functional outcomes were obtained for 16 of 22 patients. Disabilities of the Arm, Shoulder, Hand (DASH) and Short form-36 scores were collected for 14 patients who were operated on after March 2002. The mean delay from injury to surgery was 30 days (21 to 57). The mean follow-up was for 27 months (12 to 72). At the last review the mean DASH score was 14 (0 to 41). Of the 16 patients with follow-up, 13 returned to their previous employment and recreational activities without restrictions. No wound complications, infection or nonunion occurred. Malunion of the scapula can be prevented by surgical treatment of fractures in patients with delayed presentation. Surgery is safe, effective, and gives acceptable functional results.

Less Invasive Stabilization System (LISS) for fractures of the proximal tibia: indications, surgical technique and preliminary results of the UMC Clinical Trial.

The surgical treatment of proximal tibia fractures, with or without intraarticular involvement, is associated with well-described patterns of failure and significant complication rates. Recent surgical advances allow for a minimally invasive approach to such injuries, which may improve healing times, increase union rates, and decrease complication rates. The Less Invasive Stabilization System (LISS) for proximal tibia fractures employs a laterally based fixed angled implant, allowing for the placement of locking screws proximal and distal to the fracture, which may be placed percutaneously. This manuscript presents the surgical technique and indications of the Tibia LISS. A preview of the early clinical results of the treatment of bicondylar tibial plateau fractures and proximal tibia diaphysis will also be presented.

Compartment pressures after submuscular fixation of proximal tibia fractures.

INTRODUCTION: Tibial fractures are known to be associated with elevated compartment pressures, which may potentially result in a compartment syndrome. The Tibial Less Invasive Stabilization System (LISS PLT) is an internal fixator device that is designed for fixation of proximal tibia fractures. The technique includes submuscular placement of the implant along the lateral border of the tibia. Such placement of the Tibia LISS into the anterior leg compartment could increase compartment pressures, and therefore increase the risk of compartment syndrome. The objective of this prospective study is to determine whether submuscular placement of a tibia LISS fixator increases compartment pressures and what is the incidence of compartment syndrome. MATERIALS AND METHODS: Twenty-eight consecutive proximal tibia fractures were fixed using the Tibia LISS (LISS PLT). Compartment pressures in all four leg compartments were measured pre- and postoperatively. Diastolic pressures were recorded and perfusion pressures calculated. RESULTS: There were no statistically significant differences between pre- and postoperative compartment pressures. One patient out of twenty-eight developed an impending compartment syndrome after placement of the LISS implant. CONCLUSIONS: Submuscular fixation of the tibia with the Less Invasive Stabilization System does not cause a significant rise in leg compartment pressures, nor is it associated with an increased incidence of compartment syndrome.

Chromatin-dependent cooperativity between constitutive and inducible activation domains in CREB.

The cyclic AMP (cAMP)-responsive factor CREB induces target gene expression via constitutive (Q2) and inducible (KID, for kinase-inducible domain) activation domains that function synergistically in response to cellular signals. KID stimulates transcription via a phospho (Ser133)-dependent interaction with the coactivator paralogs CREB binding protein and p300, whereas Q2 recruits the TFIID complex via a direct association with hTAF(II)130. Here we investigate the mechanism underlying cooperativity between the Q2 domain and KID in CREB by in vitro transcription assay with naked DNA and chromatin templates containing the cAMP-responsive somatostatin promoter. The Q2 domain was highly active on a naked DNA template, and Ser133 phosphorylation had no additional effect on transcriptional initiation in crude extracts. Q2 activity was repressed on a chromatin template, however, and this repression was relieved by the phospho (Ser133) KID-dependent recruitment of p300 histone acetyltransferase activity to the promoter. In chromatin immunoprecipitation assays of NIH 3T3 cells, cAMP-dependent recruitment of p300 to the somatostatin promoter stimulated acetylation of histone H4. Correspondingly, overexpression of hTAFII130 potentiated CREB activity in cells exposed to cAMP, but had no effect on reporter gene expression in unstimulated cells. We propose that cooperativity between the KID and Q2 domains proceeds via a chromatin-dependent mechanism in which recruitment of p300 facilitates subsequent interaction of CREB with TFIID.

Novel mechanism of regulation of the non-receptor protein tyrosine kinase Csk: insights from NMR mapping studies and site-directed mutagenesis.

Csk (C-terminal Src kinase), a protein tyrosine kinase, consisting of the Src homology 2 and 3 (SH2 and SH3) domains and a catalytic domain, phosphorylates the C-terminal tail of Src-family members, resulting in downregulation of the Src family kinase activity. The Src family kinases share 37 % homology with Csk but, unlike Src-family kinases, the catalytic domain of Csk alone is weakly active and can be stimulated in trans by interacting with the Csk-SH3 domain, suggesting a mode of intradomain regulation different from that of Src family kinases. The structural determinants of this intermolecular interaction were studied by nuclear magnetic resonance (NMR) and site-directed mutagenesis techniques. Chemical shift perturbation of backbone nuclei (H' and (15)N) has been used to map the Csk catalytic domain binding site on the Csk-SH3. The experimentally determined interaction surface includes three structural elements: the N-terminal tail, a small part of the RT-loop, and the C-terminal SH3-SH2 linker. Site-directed mutagenesis revealed that mutations in the SH3-SH2 linker of the wild-type Csk decrease Csk kinase activity up to fivefold, whereas mutations in the RT-loop left Csk kinase activity largely unaffected. We conclude that the SH3-SH2 linker plays a major role in the activation of the Csk catalytic domain.

Site-specific incorporation of a phosphotyrosine mimetic reveals a role for tyrosine phosphorylation of SHP-2 in cell signaling.

The regulation of protein tyrosine phosphatase (PTPase) SHP-2 is proposed to involve tyrosine phosphorylation on two tail tyrosine residues. Using "expressed protein ligation", nonhydrolyzable phosphotyrosine analogs were introduced at known phosphorylation sites in SHP-2. Biochemical analysis suggests that a phosphonate at Tyr542 interacts intramolecularly with the N-terminal SH2 domain to relieve basal inhibition of the PTPase, whereas a phosphonate at Tyr-580 stimulates the PTPase activity by interaction with the C-terminal SH2 domain. Microinjection experiments indicate that a single phosphorylation of Tyr-542 of SHP-2 is sufficient to activate the MAP kinase pathway in living cells. These studies support a novel mechanism explaining how tyrosine phosphorylation of a PTPase is important in signal transduction.

Protein tyrosine kinase Csk-catalyzed phosphorylation of Src containing unnatural tyrosine analogues.

Using expressed protein ligation, five unnatural tyrosine analogues (amino-phenylalanine, homotyrosine, 2-methyl-tyrosine, (alphaS,betaR)-beta-methyl-tyrosine, and 2,6-difluoro-tyrosine) were incorporated into Src in place of the natural tail tyrosine residue. These semisynthetic substrates were evaluated as Csk substrates or allosteric activators. It appears that the tyrosine phenol hydroxyl is unlikely to be contributing significantly to Src's ground-state binding affinity for Csk. It has been observed that stabilizing tyrosine conformers can further optimize Src's already high substrate efficiency. These latter findings contrast similar studies with synthetic peptide substrates and highlight the value of investigation of protein kinase substrate selectivity with protein substrates.

Transcriptional coactivator protein p300. Kinetic characterization of its histone acetyltransferase activity.

The p300/cAMP response element-binding protein-binding protein (CBP) family members include human p300 and cAMP response element-binding protein-binding protein, which are both important transcriptional coactivators and histone acetyltransferases. Although the role of these enzymes in transcriptional regulation has been extensively documented, the molecular mechanisms of p300 and CBP histone acetyltransferase catalysis are poorly understood. Herein, we describe the first detailed kinetic characterization of p300 using full-length purified recombinant enzyme. These studies have employed peptide substrates to systematically examine the substrate specificity requirements and the kinetic mechanism of this enzyme. The importance of nearby positively charged residues in lysine targeting was demonstrated. The strict structural requirement of the lysine side chain was shown. The catalytic mechanism of p300 was shown to follow a ping-pong kinetic pathway and viscosity experiments revealed that product release and/or a conformational change were likely rate-limiting in catalysis. Detailed analysis of the p300 selective inhibitor Lys-CoA showed that it exhibited slow, tight-binding kinetics.

Bisubstrate ketone analogues as serotonin N-acetyltransferase inhibitors.

Serotonin N-acetyltransferase, also called the melatonin rhythm enzyme, is thought to play an important regulatory role in circadian rhythm in animals and people. A series of analogues were synthesized in which indole and coenzyme A were linked via ketone tethers as designed inhibitors of this enzyme. These compounds were tested against purified serotonin N-acetyltransferase. The parent ketone compound was found to be as potent as an amide linked compound studied previously, suggesting that there are no key hydrogen bonds to the nitrogen atom of the corresponding substrate necessary for tight inhibition. Reduction of the parent ketone afforded the diastereomeric carbinol mixture which showed reduced inhibitory potency, arguing against tetrahedral analogue mimicry as an important inhibitory theme. Several conformationally constrained ketone analogues were synthesized and investigated, and the results indicated that directing the orientation of the two substrates within the bisubstrate system could be used to maximize enzyme inhibition.